Project Summary/Abstract Post-traumatic stress disorder (PTSD) affects nearly 8% of the population and is more prevalent in women than men. The neurobiological factors that contribute to this sex bias are largely unknown. This project addresses this gap, in accordance with NIMH Strategic Objective 1, by determining how transcriptional regulation of memory-related genes differs in males and females in support of fear memory. Recently our group demonstrated that male and female rats differentially engage signaling mechanisms within the prefrontal cortex (PFC) during the formation of a fear memory, and our preliminary findings point to sex differences in epigenetic modification of prefrontal genes. Currently, the transcriptional regulation of episodic memory within cognitive systems is poorly understood, and the impact of sex on this dynamic process is all but unknown. This represents a significant challenge to developing effective treatment for disorders such as PTSD. The objective of this proposal is to determine how DNA hydroxymethylase TET enzymes functionally regulate fear memory formation and to identify sex-specific methylomic signatures of aversive experience. DNA 5- hydroxymethylation (5-hmC), a major regulator of active (increased) transcription in cells, has been recently implicated in fear memory formation in the brain. However, whether sex differences exist in the engagement of DNA 5-hmC mechanisms during fear memory formation remain equivocal. In our preliminary studies we found that in the rat prefrontal cortex the DNA hydroxymethylase Tet2 increased in females while Tet1 decreased in males following learning in a trace fear conditioning paradigm. The central hypothesis is that TET-mediated DNA 5-hydroxymethylation of sex-specific gene targets in the prefrontal cortex facilitates the formation of fear memory. The approach uses unbiased genome-wide analysis (NIMH Strategy 1.2, Research Priority B) in combination with viral-mediated transcriptional control to test the functional link between TET activity and memory (NIMH Strategy 1.1, Research Priority D; NIMH Strategy 1.2, Research Priority B). Aim 1 will identify the sex-specific gene targets of TET1 and TET2-mediated DNA 5-hmC in the prefrontal cortex following fear learning, using next generation whole genome chromatin immunoprecipitation (ChIP) and hydroxymethylated DNA immunoprecipitation (hmeDIP) sequencing methods. Aim 2 will use cutting-edge CRISPR-dCas9 technology to manipulate the expression of Tet1 and Tet2 in the prefrontal cortex of males and females during trace fear conditioning, which will determine the sex-dependent functional role of cortical TET1 and TET2 in fear memory formation. The proposed research is significant because it is expected to provide a (epi)genomic map of memory formation in a sexually dimorphic brain region, the medial prefrontal cortex, that is needed for understanding and even identifying new genetic biomarkers of sex-biased emotional and cognitive deficits in psychiatric illness.